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U Patented 51:13:25?

B1 activity in the living body without being aifected by 2,752,348 the vitamin B1 decomposition factors. VITAMIN B1 DERIVATIVES AND THE ANIMAL TEST PREPARATION THEREOF 5 1. Preventive effect on vitamin B1 avitaminosis of rice Taizo Matsukawa and Hajime Kawasaki, Osaka, Japan, i

assignors to Takeda Pharmaceutical Industries, Ltd., The deflvatlve Osaka, Japan No Drawing. Application January 10, 1955, Serial No. 481,013

Claims priority, application Japan June 14, 1952 8 Claims. (Cl. 260-2565) (referred to as D. A. T.), the derivative R1=CHa, R2=CsHsCO, R3=CsH5CO (referred to as D. B. T.) and vitamin B1 were administered per os to rice birds, and the changes of the body weight and survival time were recorded.

As is seen from Tables I and II the two derivatives have This invention relates to new vitamin B1 derivatives 15 the same preventive effect as vitamin B1.

TABLE I [0.02 mMol. each 01' vitamin B1 hydrochloride and D. A. '1. was administered.]

g. test 14th day 14. 1 14.1 survived. 13. 6 12.9 survived. 2 died 1 See footnote, table II.

TABLE II i fi} i it wag 4th 6th sen th 12th g day day day day day g test 14th day D. B. T. (0.02 mMol.) 13. 5 13. 4 13. 3 l3. 1 l2. 5 12. 3 l2. 4 11.91 S1211!- V V6 D. B. T. (0.06 mMol.) 13. 3 13. 2 l3. 1 13. 1 13. 2 12. 8 l2. 8 12.6 S111- vived. V. BLHCI (0.02InMoi.) 13.3 13.7 13.5 13.4 13.5 13.3 13.1 12.8 survived. Control 13.7 13.8 13.2 12.7 10.4 9.5 died 1 The control animals in Tables I and II were given food deficient in vitamin 13;.

Some other derivatives, for example those having R1=CH3, R2=CH3CO, R3=C6H5CO 0r R1=CH3, R2=C2H5CO, R3=CsH5CO, were tested with the same result.

having the general formula N=CNH2 CEO 2. Curative efiect on vitamin B1 avitaminosis of pigeons and rice birds (1) Twenty 'y of D. A. T. was injected into breast muscle of each of three rice birds, and a small quantity of D. B. T. was administered per os to each of other three rice birds, all suliering from severe vitamin B1 Derivatives M. P. (with decomp.) Solvent for recrystallization ag R1 R, H:

CH;- CH;GO OH;CO 122i23 C A mixture of benzene and benzine Colin-less pr sms. 011,- CQHgOO- OH5OO 173174 C Dilute alcohol Do. CHrp-NO1CuH4CO- H 135 C Water D0. CH; OH;O 0 OtH5OO-- Lil-142 0 A mixture of benzene and benzine Do. CH; CgHaCO- C5H500 134-135 0 do D0. CH; G3H7CO- OQH5CO- 107108 O .d0. D0. OH;- p-NO:O H4O0 CQHSOO- 169170 0 Alcohol. Do. CHg- 0,11500- CH5CO 104105 C Ether Do. 0111- C;H1C 0- 01310 0- (SQ- C A mixture of benzene and benzine. Do. CH; CxH7CO- l29130 0.- Benzene Do. CH;- CaHs0.00 051150 O- Lil-142 Dilute methanol Do. C2H5- CH;CO CH;CO 113114 C A mixture of benzene and benzine Do. CzH5 CtHgC 0- 011E500. 171173 C. (hydro A mixture of alcohol and ether Do. OH;- CHaCO- CBH5CO 210211 C. (perchloric acid salt) Dilute alcohol Do. (1H;- CuHiC O. OH5CO- 165 C. (nitric acid salt)--. Water D0. CH; CtH G O- CgHgCO- 7880 O. (hydrobromide) do O 010 (glles s nee es OH;- C|H5OO- CHiOO Sit- O. (sulfuric acid salt) do. Do.

These derivatives are thought to be absorbed from the avitaminosis. In all cases the symptom was alleviated intestinal canal to liberate vitamin B1 and display vitamin appreciably after one hour except one case.

(2) Curative daily dose of vitamin B1 hydrochloride and D. A. T. was tested on the typical polyneuritis of pigeons. in all cases the symptom was alleviated within several hours.

From the above results it is evident that D. A. T. has the same curative effect as vitamin B1.

3. Test of toxicity The toxicity of D. A. T. was tested according to the Behrens-Korber method, using mice as test animals. LDso of D. A. T. administered by intravenous injection was 0.0104 mMol/lO g. and that of vitamin B1 hydrochloride was 0.00207 mMol/ g. That is, the toxicity of the former is one fifth of that of the latter.

4. Test of excretion in urine after large dose administration TABLE III Amount of vitamin B! excreted within 3 hours Vitamin B1.HCl O.47:l:0.16 mg. D. A. T 2.58:l:(l.89 mg. D. B. T 1.80i0.98 mg.

As is evident from the table D. A. T. is excreted in the largest quantity and it is clear that D. A. T. and D. B. T. are absorbed more readily than vitamin B1.

The preparation of the new derivatives of vitamin B1 is described in the following:

In solution vitamin B1 and its derivatives exist in a state of equilibrium between ammonium-type and thioltype, and this equilibrium shifts to the ammonium-type in an acid medium and to the thiol-type in an alkaline medium. Since the compounds of the present invention are derivatives of the thiol-type vitamin B1, the object of the present invention is attained profitably by acylating vitamin B1 or its derivatives at a pH above 7. The reaction can be conducted in a medium of a pH higher than 10, so long as the material is not decomposed in this state. In general the solution of vitamin B1 or its derivatives in a strong alkaline medium is stable at room temperature. On the other hand, the yield of the reaction effected in a medium of a pH below 7 is poor, because the thiol-type of vitamin B1 or its derivatives exists in small quantity in this state.

The reaction of the present invention is represented by the following equation:

l Ha. CHr-CHg-O-Rs wherein R1 is a methyl or ethyl group, R2 an acyl group and R3 hydrogen or an acyl group.

In this reaction acid anhydrides, acid halides or other acylating agents are employed as acylating agent. Illustrative of the acyl group to be introduced are, for example, acetyl, propionyl, butyryl, benzoyl, p-nitrobenzoyl, carbalkoxyl and other aliphatic and aromatic acyl groups with or Without substituent.

The reaction is carried out advantageously in water, alcohol, methanol, pyridine or other suitable solvent, "so long as the latter does not affect the reaction.

Depending on the acylating agent, the acyl group is introduced only in the sulfhydryl group (-SH) or in both the sult'hydryl and hydroxyl group of the hydroxyethyl group. That is, the acyl group is generally introduced first into the sulfhydryl group, then into the hydroxyl group, but if the S-acyl compound is sparingly soluble in the solvent its further acylation stops. These compounds react with inorganic or organic acids to yield their salts. To take hydrochloride as an example, thioltype vitamin B1 diacetyl, dibenzoyl and dipropionyl derivatives give their hydrochlorides in colorless prisms melting at ll9l20 C. (decomp), 160 C. (decomp.) and 188l89 C. (decomp), respectively.

These hydrochlorides are more stable to heat than their free bases. For instance, when a solution in water of thiol-type vitamin B1 diacetyl derivative hydrochloride and that of its free base are heated at C. for 30 minutes, subjected separately to partition chromatography and sprayed with the Dragendorff reagent, the latter reveals decomposition products while the former does not. All of the products have the same activity as vitamin B1, are not decomposed by the vitamin B1 decomposition factors and are absorbed effectively from the intestinal canal when administered per 0s.

The said compounds also form other therapeutically applicable, i. e. non-toxic, acid salts, as for example other hydrohalides than the hydrochloride, e. g. the hydrobromide, hydroiodide, etc., and the sulfate, nitrate, phosphate, perchlorate, thiocyanate, acetate, propionate, oxalate, malate, succinate, tartrate, citrate, benzoate, methane-sulfonate, ethane-sulfonate, benzene-sulfonate, toluene-sulfonate, etc.

Representative illustrative embodiments of the invention are set forth in the following examples. In these examples, the parts and percentages are by weight.

Example 1.To a solution of 2 parts of vitamin B1 hydrochloride in 5 parts of water, there are added 7 parts of an aqueous sodium hydroxide solution of 10% strength followed dropwise by 1.5 parts of acetic anhydride with stirring. During the reaction which ensues, the mixture is kept alkaline by the occasional addition of aqueous sodium hydroxide solution. Sodium chloride is then added to the reaction mixture to salt out the product, which is recrystallized from a mixture of benzene and petroleum benzine, in the form of colorless prisms melting at' l22l23 C., with decomposition. The yield is 1.7 parts.- The product, which is readily soluble in water, alcohol,

acetone and ethyl acetate, fairly soluble in ether and-- benzene, and sparingly soluble in petroleum benzine, has a structure represented by the formula The product forms a hydrochloride which melts at ll)- C. (with decomposition).

Example 2.To 'a solution of 1 part of vitamin Br I less prisms melting at 173-174 C. with decomposition. The yield is 0.8 part. The product which is readily soluble in alcohol and acetone and sparingly soluble in water, has a structure represented by the formula Example 3.To a solution of 2 parts of vitamin B1 hydrochloride in 5 parts of water, there are added 7 parts of aqueous sodium hydroxide solution of strength, followed dropwise by a solution of 1.2 parts of p-nitrobenzoylchloride in 2 parts of ethyl acetate with stirring. During the reaction which ensues, the mixture is kept alkaline by the occasional addition of aqueous sodium hydroxide solution. The product is filtered, dissolved in dilute hydrochloric acid, and the solution neutralized by sodium bicarbonate to precipitate the product in the form of colorless prisms melting at 135 C. with decomposition. The yield is 2 parts. The product which is sparingly soluble in water and organic solvents has a structure represented by the formula Example 4.To a solution of 3 parts of benzoyl vitamin B hydrobromide in 20 parts of water, there are added 7 parts of an aqueous sodium hydroxide solution of 10% strength, followed dropwise by 1 part of acetic anhydride with stirring, whereupon an oily product separates which solidifies immediately. During the reaction the mixture is kept alkaline by the occasional addition of aqueous sodium hydroxide solution. The product is recrystallized, from a mixture of benzene and petroleum benzine, in the form of colorless prisms melting at 141-142 C. with decomposition. The yield is 1.7 parts. The product has a structure represented by the formula Example 5.To a solution of 6 parts of benzoyl vitamin B; hydrobromide in 50 parts of water, there are added 14 parts of aqueous sodium hydroxide solution of 10% strength, followed dropwise by 1.6 parts of propionic anhydride with stirring, whereupon an oily product separates which solidifies immediately. During the reaction the mixture is kept alkaline by the occasional addition of sodium hydroxide solution. The product is recrystallized, from a mixture of benzene and petroleum benzine, in the form of colorless prisms melting at 134-135 C. with decomposition. The yield is 4 parts. The product has a structure represented by the formula CHO Example 6.To a solution of 2.5 parts of benzoyl vitamin B1 hydrobromide in 10 parts of water, there are added 7 parts of aqueous sodium hydroxide solution of 10% strength followed dropwise by 1 part of butyric anhydride With stirring, whereupon an oily product separates which solidifies immediately. During the reaction the mixture is kept alkaline by the occasional addition of sodium hydroxide solution. The product is recrystallized, from a mixture of benzene and petroleum benzine, in the form of colorless prisms, melting at 107-108 C. with decomposition. The yield is 1.5 parts. The product has a structure represented by the formula of 10% strength followed dropwise by 1 part of benzoyl chloride with stirring whereupon an oily product separates which solidifies immediately. During the reaction, the mixture is kept alkaline by the occasional addition of aqueous sodium hydroxide solution. The product is recrystallized, from dilute alcohol, in the form of colorless prisms melting at 173174 C. with decomposition. The yield is 2.2 grams. The product has a structure represented by the formula N=c-Nrn 0110 CHa( 3 --C zN lm-aH C=CS OC CBHIS H; HzCHz-OOC.COH5 The product forms a hydrochloride which melts at 160 C. with decomposition.

Example 8.To a solution of 3 parts of benzoyl vitamin B1 hydrobromide in 30 parts of water, there are added 7 parts of aqueous sodium hydroxide solution of 10% strength followed dropwise by a solution of 1.2 parts of p-nitrobenzoyl chloride in 10 parts of ethyl acetate. During the reaction which ensues, the mixture is kept alkaline by the occasional addition of aqueous sodium hydroxide solution. The product which separates is recrystallized from alcohol in the form of colorless prisms melting at 169-170 C. with decomposition. The yield is 2.3 parts. The product has a structure represented by the formula Example 9.To a solution of 2 parts of vitamin B1 hydrochloride in 10 parts of water, there are added 7 parts of an aqueous sodium hydroxide solution of 10% strength. To the resultant solution, after saturation- The product forms a hydrochloride which melts at 188- 189 C. with decomposition.

Example 10.To a solution of 3 parts of benzoyl vitamin B1 hydrobromide in 15 parts of water, there is added a solution of 0.8 part of sodium hydroxide in 5 parts of water. After the resultant solution has been allowed to stand-for an hour there is added thereto portionwise 0.7 party of ethyl chlorocarbonate while cooling with ice and stirring vigorously, whereupon a viscous oil separates which is crystallized by triturating with parts of ether. The product is recrystallized from dilute methanol, in

7 the form of colorless prisms, melting at 141-142" C. The yield is 2 parts. The product has a structure represented by the formula Example I1.-To a solution of 2 parts of vitamin B1 hydrochloride in parts of water, there are added 7 parts of aqueous sodium hydroxide solution of 10% strength followed dropwise by 2 parts of butyric anhydride While stirring, whereupon an oil product separates which solidifies gradually. During the reaction, the mixture is kept alkaline by the occasional addition of aqueous sodium hydroxide solution. The product is recrystallized from a mixture of benzene and benzine in the form of prisms melting at 6970 C. The yield is 1.5 parts. The product has a structure represented by the formula Example 12.To a solution of 1 part of the hydrochloride of homovitamin B1 (a compound having the same structure as vitamin B1 except that the methyl group at C2 of the pyrimidine nucleus is replaced by an ethyl group) in 7 parts of Water, there are added 3.5 parts of aqueous sodium hydroxide solution of 10% strength. To the resultant solution, after saturation thereof with sodium chloride, there is added, dropwise, and while stirring 1 part of acetic anhydride, whereupon an oily prod uct separates which solidifies gradually. During the reaction, the mixture is kept alkaline by the occasional addition of sodium hydroxide solution. The product is recrystallized, from a mixture of benzene and petroleum benzine, in the form of colorless prisms melting at 113 114 C. with decomposition. The yield is 0.8 part. The product has a structure represented by the formula Example 13.-To a solution of 1 part of homovitamin B1 hydrochloride in parts of water, there are added 3.5 parts of aqueous sodium hydroxide solution of 10% strength, followed dropwise and while stirring by 1 part of benzoyl chloride, whereupon an oily product separates. During the reaction, the mixture is kept alkaline by the occasional addition of aqueous sodium hydroxide solution. he product is dissolved in ethyl acetate, and the solution is dried with anhydrous sodium sulfate and then evaporated to dryness in vacuo. The residue is dis solved in a small quantity of absolute alcohol, after which there is added alcohol containing hydrochloric acid and then a large quantity of ether to separate crystals of the hydrochloride. The product is recrystallized from a mixture of alcohol and ether, in the form of colorless prisms melting at 171473 C. with decomposition. The yield is 0.8 part. The product has a structure represented by the formula hol, whereupon it has a melting point of 210-211 C.,

with decomposition. The salt corresponds to the formula N=O--NHs.HClOi CHO Example 15.--Dilute nitric acid is added to the base obtained according to Example 7, whereupon there is obtained a syrupy product which soon solidifies. The thus-obtained nitrate is recrystallized from water whereupon it melts at C. with decomposition. The nitrate corresponds to the formula Example 17.Dilute sulfuric acid is added to the base obtained according to Example 7, whereupon a syrupy product is obtained which soon solidifies. The resultant sulfate is purified by recrystallization from water, whereupon it melts at 83-85" C. The product has two mols of water of crystallization; it corresponds to the formula Example 18.-To a solution of 2 parts of vitamin B1 hydrochloride in 10 parts of Water, there are added 7 parts of an aqueous "sodium hydroxide solution of 10% strength. To the resultant solution, after saturation thereof with sodium chloride, there is added dropwise, while stirring, 1 part of butyric anhydride, whereupon an oily product separates. During the reaction, the mixture is kept alkaline by the occasional addition of aqueous sodium hydroxide solution. The product is dissolved in ethyl acetate and the solution is dried in vacuo. The residue is recrystallized from benzene in the form of colorless prisms melting at 129-130 C. with decomposi- CH3 CHr-CHr-OH This application is in part a continuation of copending application Serial No. 358,965, filed June 1, 1953.

Having thus disclosed the invention, what is claimed is:

1. A member selected from the group consisting of compounds having the general formula and their therapeutically useful acid salts, wherein R1 is *a member selected from the group consisting of methyl 9 and ethyl radicals, R2 is a member selected from the group consisting of lower fatty acid acyl radicals and mononuclear aryl carboxylic acid acyl radicals and R3 is a member selected from the group consisting of hydrogen, lower fatty acid acyl radicals and mononuclear aryl carboxylic acid acyl radicals.

2. Compound having the formula i i-H o=c-s-0 0-0411:

Ha Hr-CHaOOC.CeHa 5. Compound having the formula N=CNH2.HC1 CHO -CH2N C=CS-OC.CHI

Ha Ha-CHa-O-O C-CH: 6. Compound having the formula N=CNHg-HC1 CHO CHr-C CH|N 1 1' H C=O-SOC.CH5

H: Hz-CHrO-OC.COH

7. A process for preparing the compounds having the general formula at nonacidic pH, with a member selected from the group consisting of carboxylic acid anhydrides and carboxylic acid halides.

8. A process for preparing the compounds having the general formula wherein R1 represents a member selected from the group consisting of methyl and ethyl radicals, which comprises reacting the compounds having the general formula N=?NH1 /CHS Rr- O-CHY-N Ha fiz-CHrOH at nonacidic pH, with p-nitrobenzoyl chloride.

References Cited in the file of this patent FOREIGN PATENTS 1,068,459 France June 25, 1954 

1. A MEMBER SELECTED FROM THE GROUP CONSISTING OF COMPOUNDS HAVING THE GENERAL FORMULA
 7. A PROCESS FOR PREPARING THE COMPOUNDS HAVING THE GENERAL FORMULA 